Xanthine oxidase of Acinetobacter calcoaceticus RL2-M4: Production, purification and characterization.

Xanthine oxidase (EC 1.17.3.2) is a key enzyme of purine metabolism and has potential applications in food and pharmaceutical industries. In the present study, a new bacterial source of xanthine oxidase i.e. Acinetobacter calcoaceticus RL2-M4 with high oxidase activity was isolated from soil. The culture conditions were optimized with one variable at a time (OVAT) and response surface methodology (RSM) approaches included: a minimal salt medium (MSM) of pH 7.0 supplemented with 0.8% yeast extract, 8.5 mM xanthine and incubation at 30 °C for 24 h. Under these culture conditions 11.57 fold increase in the production of this enzyme was achieved. The enzyme was purified from A. calcoaceticus RL2-M4 using anion exchange chromatography to 8.18 fold with 31% yield and specific activity of 4.58 U/mg protein. SDS-PAGE analysis of the purified enzyme revealed that it was homodimer of 95 kDa and its native molecular mass was estimated to be 190 kDa. This enzyme was found to be stable at 35 °C for 5 h. The purified xanthine oxidase of A. calcoaceticus RL2-M4 had Km 0.3 mM and Vmax 5.8 U/mg protein using xanthine as substrate. The activity and stability characteristic of xanthine oxidase of A. calcoaceticus RL2-M4 makes it a potentially good enzyme for industrial applications.

Potential cow milk xanthine oxidase inhibitory and antioxidant activity of selected phenolic acid derivatives.

Xanthine oxidase (XO) found in all mammals and excess activity leads to urolithiasis. The cow milk XO was purified to 305-fold with a specific activity of 8.76 EU/mg and overall yield of 87% by using DEAE-Sepharose chromatography. The phenolics showed potent XO inhibitory effect with Ki , P1 (0.412), P2 (0.632), P3 (0.585), P4 (0.886), P5 (1.633), P6 (0.503), P7 (2.882), P8 (3.761), P9 (4.487), and P10 (5.841) µM. The phenolics P9 and P10 exhibited uncompetitive inhibition; the phenolics P1, P2, P3, P4, and P6 showed competitive inhibition, and other phenolic acids showed noncompetitive inhibition. The studied phenolic compounds showed potent antioxidant activity and expressed as EC50 , ranged from, DPPH (4.2-25.8 µg mL-1 ), ABTS (10.2-42.5 mmol TE 100 g-1 ), and FRAP (6.3-36.8 mol Fe (II) 100 g-1 ). The results obtained from this study might be utilized for design of XO inhibitors and as antigout agent.

New insights into xanthine oxidase behavior upon heating using spectroscopy and in silico approach.

Thermal dependent conformational changes of xanthine oxidase (XOD) were studied using sensitive and non-destructive methods like fluorescence spectroscopy and molecular modeling in the temperature range of 25-85°C. Intrinsic fluorescence studies showed that the microenvironment of tryptophan and tyrosine residues becomes more exposed to solvent as the temperature increased up to 85°C, whereas in case of flavin cofactor is rather conserved. At higher temperatures, the flavin adenine dinucleotide is displaced from the core of the protein, but is not fully released as shown by the Stern Volmer quenching constant and accessible fraction of the cofactor. Anyway, no significant changes in the structure of XOD monomer were identified after running molecular dynamics simulations at temperatures 25°C, 65°C and 85°C. Therefore, we can conclude that the most important changes in the protein structure at thermal treatment mainly consist on molecular aggregation and dissociation events.

Human xanthine oxidase recombinant in E. coli: A whole cell catalyst for preparative drug metabolite synthesis.

Human xanthine oxidoreductase (XOR), which is responsible for the final steps of the purine metabolism pathway and involved in oxidative drug metabolism, was successfully expressed in Escherichia coli BL21(DE3) Gold. Recombinant human (rh) XOR yielded higher productivity with the gene sequence optimized for expression in E.coli than with the native gene sequence. Induction of XOR expression with lactose or IPTG resulted in complete loss of activity whereas shake flasks cultures using media rather poor in nutrients resulted in functional XOR expression in the stationary phase. LB medium was used for a 25L fermentation in fed-batch mode, which led to a 5 fold increase of the enzyme productivity when compared to cultivation in shake flasks. Quinazoline was used as a substrate on the semi-preparative scale using an optimized whole cell biotransformation protocol, yielding 73mg of the isolated product, 4-quinazolinone, from 104mg of starting material.

Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel.

A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150 kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60 °C and incubated for 60 min. These results indicated that the enzyme was heat stable.

Novel affinity purification of xanthine oxidase from Arthrobacter M3.

An affinity protocol for purification of xanthine oxidase (XOD) from Arthrobacter M3 was developed. The isolation procedure consisted of only three steps, ammonium sulfate precipitation, affinity extraction to exclude the major impurities, and the final refining procedure with DEAE ion-exchange chromatography for removal of minor contaminants. In this affinity preparation, guanine, an analogue of xanthine, was chosen as the affinity ligand, and was coupled with Sepharose 4B through spacers composed of epichlorohydrin and ethylenediamine. Crude protein has been run through ammonium sulfate precipitation and the affinity column, 99.1% of proteins were removed. After DEAE ion-exchange chromatography, the purity of the refined XOD was 97.5% by Native-PAGE analysis. The activity recovery of purified XOD (36.1%) was almost higher than that of other methods reported. Reducing SDS-PAGE analysis showed that the purified XOD (one band in Native-PAGE analysis) showed two polypeptides with the molecular weights ∼35kDa and ∼100kDa, respectively. The desorption constant K(d) and the theoretical maximum absorption Q(max) on the affinity medium were 3.0µg/ml and 2.2mg/g medium in absorption analysis.

Preparation and characterization of affinity sorbents based on isoalloxazine-like ligands for separation of flavoenzymes.

Affinity ligands for flavoenzymes were synthesized based on the natural structure of flavo-coenzymes. Two typical flavoenzymes, cholesterol oxidase from Brevibacterium sp. and xanthine oxidase from bovine milk, were employed as standard enzymes. Fluorescent probes were synthesized from eight isoalloxazine-like chemicals and 5-aminofluorescein. Probe-enzyme interactions were analyzed via fluorescence spectra. Chemicals with high binding abilities to flavoenzymes were coupled with Sepharose through spacers composed of epichlorohydrin, ethylenediamine, 1,3-diaminopropane, 2-hydroxy-1,3-diaminopropane, and 1,4-diaminobutane, and subjected to adsorption analysis with flavoenzymes. The results indicated that ligands synthesized from 2,4-dioxohexahydropyrimidine-5-carboxylic acid, cytosine, 7-chloroalloxazine, and 8-chloroalloxazine had high binding abilities to the flavoenzymes. The affinity sorbent based on these ligands revealed a high theoretical maximum adsorption (Q(max)). Protein and bioactivity recoveries were tested after one step of affinity binding via chromatographic analysis on small columns. Results showed that ligands linked with sorbents through long hydrophilic spacers had higher activity recoveries.

Substrate Orientation and Catalysis at the Molybdenum Site in Xanthine Oxidase: CRYSTAL STRUCTURES IN COMPLEX WITH XANTHINE AND LUMAZINE.

Xanthine oxidoreductase is a ubiquitous cytoplasmic protein that catalyzes the final two steps in purine catabolism. We have previously investigated the catalytic mechanism of the enzyme by rapid reaction kinetics and x-ray crystallography using the poor substrate 2-hydroxy-6-methylpurine, focusing our attention on the orientation of substrate in the active site and the role of Arg-880 in catalysis. Here we report additional crystal structures of as-isolated, functional xanthine oxidase in the course of reaction with the pterin substrate lumazine at 2.2 A resolution and of the nonfunctional desulfo form of the enzyme in complex with xanthine at 2.6 A resolution. In both cases the orientation of substrate is such that the pyrimidine subnucleus is oriented opposite to that seen with the slow substrate 2-hydroxy-6-methylpurine. The mechanistic implications as to how the ensemble of active site functional groups in the active site work to accelerate reaction rate are discussed.

Physicochemical and kinetic properties of purified sheep's milk xanthine oxidoreductase.

Xanthine oxidoreductase (XOR) was purified for the first time from sheep's milk. The ultraviolet-visible absorption spectrum was essentially identical to those of the corresponding bovine, human, and goats' milk enzymes and showed an A280/A450 ratio of 5.35 +/- 0.24, indicating a high degree of purity. Like milk XOR from other species, sheep's milk enzyme showed a single band on SDS-PAGE corresponding to a subunit with approximate Mr 150,000. Xanthine oxidase activity of purified sheep's milk XOR (0.69 +/- 0.04 micromole urate min(-1) mg(-1)) was low relative to that of the bovine milk enzyme (1.83 +/- 0.02 micromole urate min(-1) mg(-1)), but higher than those of human or goats' milk XOR. As in the latter 2 cases, the low activity of sheep's milk XOR can be attributed to its relatively low molybdenum content (0.18 atoms per subunit), compared with that of the bovine milk enzyme (0.56 atoms Mo per subunit). Consistent with this, NADH oxidase activity of sheep's milk XOR was similar to that of enzymes purified from bovine, human, or goats' milk. The presence of desulpho-enzyme in sheep's milk XOR was demonstrated by resulfuration experiments, whereby xanthine oxidase activity was increased by approximately 75%.

Purification and partial characterisation of camel milk xanthine oxidoreductase.

Xanthine oxidoreductase (XOR) was purified in the presence of dithiothrietol from camel milk with yields of up to 22.2mg/l that were comparable to those obtained from bovine and human milk sources. On SDS-PAGE, the freshly purified camel milk XOR had a protein flavin (A280/A450) ratio of 5.3 +/- 0.4 and appeared homogenous with a single major band of approximately Mr 145.3 KDa. Surprisingly, in all the batches (n = 8) purified camel milk XOR showed no detectable activity towards xanthine or NADH. The molybdenum content of camel XOR was comparable to human and goat milk enzymes. After resulphuration, camel milk XOR gave a specific activity of 1.1 nmol/min/mg and 13.0 nmol/min/mg enzyme towards pterin (fluorimetric assay) and xanthine (spectrophotometric assay) respectively. This activity was markedly lower than that of human, bovine and goat enzymes obtained under the same conditions. These findings suggest that the molybdo-form of camel enzyme is totally under desulpho inactive form. It is possible that camel neonates are equipped with an enzymic system that reactivates XOR in their gut and consequently generates antibacterial reactive oxygen species.

Peroxisomes from pepper fruits (Capsicum annuum L.): purification, characterisation and antioxidant activity.

Pepper is a vegetable of importance in human nutrition. Currently, one of the most interesting properties of natural products is their antioxidant content. In this work, the purification and characterisation of peroxisomes from fruits of a higher plant was carried out, and their antioxidative enzymatic and non-enzymatic content was investigated. Green and red pepper fruits (Capsicum annuum L., type Lamuyo) were used in this study. The analysis by electron microscopy showed that peroxisomes from both types of fruits contained crystalline cores which varied in shape and size, and the presence of chloroplasts and chromoplasts in green and red pepper fruits, respectively, was confirmed. Peroxisomes were purified by differential and sucrose density-gradient centrifugations. In the peroxisomal fractions, the activity of the photorespiration, beta-oxidation and glyoxylate cycle enzymes, and the ROS-related enzymes catalase, superoxide dismutase, xanthine oxidase, glutathione reductase and NADP(+)-dehydrogenases, was determined. Most enzymes studied had higher specific activity and protein content in green than in red fruits. By native PAGE and western blot analysis, the localisation of a Mn-SOD in fruit peroxisomes was demonstrated. The ascorbate and glutathione levels were also determined in crude extracts and in peroxisomes purified from both green and red peppers. The total ascorbate content (200-220 mg per 100 g FW) was similar in crude extracts from the two types of fruits, but higher in peroxisomes from red peppers. The glutathione concentration was 2-fold greater in green pepper crude extracts than in red fruits, whereas peroxisomes from both tissues showed similar values. The presence in pepper peroxisomes of different antioxidative enzymes and their corresponding metabolites implies that these organelles might be an important pool of antioxidants in fruit cells, where these enzymes could also act as modulators of signal molecules (O2*-, H202) during fruit maturation.

Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system.

cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.

Purification, crystallization and preliminary X-ray diffraction studies of xanthine dehydrogenase and xanthine oxidase isolated from bovine milk.

Xanthine dehydrogenase catalyzes the oxidation of hypoxanthine to xanthine and the further oxidation of xanthine to uric acid. The enzyme is the target of the anti-gout drug allopurinol and its involvement in postischemic reperfusion injury is presently being defined. Each subunit of the homodimeric 290 kDa enzyme contains four cofactors: one Mo-pterin, two [2Fe-2S] clusters and one FAD. Both the dehydrogenase (XDH) and the proteolytically modified oxidase form (XO) of the enzyme from bovine milk have been crystallized. XO crystals belong to space group C222(1), with unit-cell parameters a = 116.3, b = 164.4, c = 153.2 A at room temperature and a = 117.8, b = 165.4, c = 154.5 A when flash-frozen. They allow data collection to 3.3 and 2.5 A, respectively. In addition, a data set was collected from frozen XDH crystals and processed to 2.1 A. These crystals belong to space group C2, with unit-cell parameters a = 169.9, b = 124.8, c = 148.6 A, beta = 90.9 degrees. The unit-cell volumes and Matthews parameters are similar for the two crystal forms. There is one monomer per asymmetric unit in the XO crystals and a complete native dimer per asymmetric unit in the XDH crystals.

Antibacterial properties of xanthine oxidase in human milk.

Formula-fed babies contract gastroenteritis more than breast-fed babies, which is of concern to mothers who cannot breastfeed or, as with HIV-infected mothers, are discouraged from breastfeeding. The ability of endogenous breastmilk xanthine oxidase to generate the antimicrobial radical nitric oxide has been measured and its influence on the growth of Escherichia coli and Salmonella enteritides examined. Breastmilk, but not formula feed, generated nitric oxide. Xanthine oxidase activity substantially inhibited the growth of both bacteria. An important natural antibiotic system is missing in formula feeds; the addition of xanthine oxidase may improve formula for use when breastfeeding is not a safe option.

Mouse mammary gland xanthine oxidoreductase: purification, characterization, and regulation.

Xanthine oxidoreductase (XOR) has been purified from lactating mouse mammary tissue and its properties and developmental expression have been characterized. XOR was purified 80-fold in two steps using benzamidine-Sepharose affinity chromatography. The purified enzyme had a specific activity of 5.7 U/mg and an activity to flavin ratio of 192. SDS-polyacrylamide gel electrophoresis showed that it was composed of a single (150 kDa) band and N-terminal sequence analysis verified that it was intact mouse XOR. Isoelectric focusing showed that purified XOR was composed of three catalytically active, electrophoretic variants with pI values of 7.55, 7.65, and 7.70. The majority of the XOR activity in both pregnant and lactating mammary glands was shown to exist as NAD+-dependent dehydrogenase (XD form), while the enzyme in freshly obtained mouse milk exits as O2-dependent oxidase (XO form). The activity and protein levels of XOR selectively increased in mammary tissue during pregnancy and lactation. The time course of these increases was biphasic and correlated with the functional maturation of the mammary gland. These results indicate that XOR may have novel, mammary gland-specific functions, in addition to its role in purine metabolism.

Simple, high-yield purification of xanthine oxidase from bovine milk.

Xanthine oxidase, a commercially important enzyme with a wide area of application, was extracted from fresh milk, without added preservatives, using toluene and heat. The short purification procedure, with high yield, consisted of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast flow) column chromatography. Xanthine oxidase was eluted as a single activity peak from the column using a buffer gradient. The purification fold, specific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in specific activity was observed. Activity and protein gave coincident staining bands on native polyacrylamide gels. The intensity and the number of bands were dependent on the oxidative state(s) of the enzyme; reduction by 2-mercaptoethanol decreased the intensity of the slow-moving bands and increased the intensity of the fastest-moving band. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for > or = 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purified xanthine oxidase becomes a heterodimer due to endogenous proteases.

Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range.

Modulation of renal xanthine oxidoreductase in aging: gene expression and reactive oxygen species generation.

Xanthine oxidoreductase (XD: xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes oxidation of hypoxathine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XOD). XOD is one of the major cellular sources of superoxide production and is well known as a causative factor in ischemia/reperfusion damage. At present, almost no information on the conversion status is available with respect to aging. In the present study, we investigated the effect of age on the XOD/XDH status and gene expression in the kidney. In addition, we assessed XOD-induced reactive oxygen species (ROS) using the dichlorofluoroscein (DCF) method. Our results show that XD activity gradually up to 18 months of age and then a slight decrease at 24 months of age. XDH activity showed increases up to 18 months of age, then decreased at 24 months of age. The conversion of XDH to XOD, assessed by changes in the ratios of XOD/(XOD+XDH), showed an age-related increase, which peaked at 24 months. Levels of XD protein and its mRNA paralleled to overall XD activity. ROS generation has tendency to increase with age. Our results suggest that the increased conversion of XDH to XOD observed with age may be an important contributing factor to the increased renal oxidative stress during aging.

Solubilization and purification of xanthine oxidase from bovine milk fat globule membrane.

Bovine milk xanthine oxidase (XO) was isolated and purified from milk fat globule membrane (MFGM). The method included the following steps: solubilization of XO from MFGM in 200 mM dithiothreitol (DTT) at pH 8.0, fractionation of solubilized proteins with ammonium sulfate, chromatography on DEAE-Sepharose with gradient elution, and rechromatography of the XO fraction for final purification. The method is highly reproducible, is comparatively simple, and provides highly pure enzyme. Purified XO, analyzed by (8%) SDS-PAGE, had only one band of 140-150 kDa. XO showed a high specific activity of 2.5 units/mg of protein and an A280: A450 ratio of 4.8.